U.S. patent application number 10/538590 was filed with the patent office on 2008-02-07 for sequences diagnostic for foot and mouth disease.
Invention is credited to Linda J. DeCarolis, Richard C. Ebersole, Raymond E. Jackson.
Application Number | 20080032285 10/538590 |
Document ID | / |
Family ID | 32682133 |
Filed Date | 2008-02-07 |
United States Patent
Application |
20080032285 |
Kind Code |
A1 |
Ebersole; Richard C. ; et
al. |
February 7, 2008 |
Sequences Diagnostic for Foot and Mouth Disease
Abstract
Methods and materials for the detection of the foot and mouth
disease virus (FMDV). The methods may utilize PCR amplification,
with or without an internal positive control, and appropriate
primer pairs. The reagents to perform these methods can be supplied
as a kit and/or in tablet form.
Inventors: |
Ebersole; Richard C.;
(Wilmington, DE) ; DeCarolis; Linda J.; (Newark,
DE) ; Jackson; Raymond E.; (Newark, DE) |
Correspondence
Address: |
E I DU PONT DE NEMOURS AND COMPANY;LEGAL PATENT RECORDS CENTER
BARLEY MILL PLAZA 25/1128, 4417 LANCASTER PIKE
WILMINGTON
DE
19805
US
|
Family ID: |
32682133 |
Appl. No.: |
10/538590 |
Filed: |
December 19, 2003 |
PCT Filed: |
December 19, 2003 |
PCT NO: |
PCT/US03/41808 |
371 Date: |
July 30, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60434974 |
Dec 20, 2002 |
|
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|
Current U.S.
Class: |
435/6.14 ;
435/6.16; 536/23.1 |
Current CPC
Class: |
C12N 2770/32111
20130101; C12Q 1/6883 20130101; C07H 21/04 20130101 |
Class at
Publication: |
435/6 ;
536/23.1 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07H 21/00 20060101 C07H021/00 |
Claims
1. A method for detecting the presence of FMDV in a sample, the
method comprising: (a) performing RT-PCR amplification of the
sample using at least one primer pair selected from the group
consisting of: (i) SEQ ID NOs:16 and 17, (ii) SEQ ID NOs:16 and 18,
(iii) SEQ ID NOs:16 and 19, and (iv) SEQ ID NOs:16 and 20, to
produce an RT-PCR amplification result; and (b) examining the
RT-PCR amplification result of step (a) to detect for an
amplification product of the primer pair, whereby a positive
detection of the amplification product indicates the presence of
FMDV in the sample.
2. The method of claim 1, wherein in step (b) a melting curve
analysis is used to detect for an amplification product.
3. The method of claim 1, further comprising a step of extracting
RNA from the sample prior to said step (a).
4. An isolated polynucleotide for detection of FMDV comprising SEQ
ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID
NO:20.
5. A kit for detection of FMDV, comprising: (a) at least one primer
pair selected from the group consisting essentially of: (i) SEQ ID
NOs:16 and 17, (ii) SEQ ID NOs:16 and 18, (iii) SEQ ID NOs:16 and
19, and (iv) SEQ ID NOs:16 and 20; (b) reverse transcriptase; and
(c) thermostable DNA polymerase.
6. A replication composition for use in performance of RT-PCR,
comprising: (a) at least one primer pair selected from the group
consisting essentially of: (i) SEQ ID NOs:16 and 17, (ii) SEQ ID
NOs:16 and 18, (iii) SEQ ID NOs:16 and 19, and (iv) SEQ ID NOs:16
and 20; (b) reverse transcriptase; and (c) thermostable DNA
polymerase.
7. A tablet comprising the replication composition of claim 6.
8. A kit for detection of FMDV in a sample, comprising the tablet
of claim 6.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/434,974, filed Dec. 20, 2002, the entire
contents of which is hereby incorporated by reference in its
entirety.
FIELD OF THE INVENTION
[0002] The field of invention relates to diagnostic testing, and
more specifically, to diagnostic methods and materials for
detecting the Foot and Mouth Disease Virus (FMDV).
BACKGROUND OF THE INVENTION
[0003] Recent events in the United Kingdom have demonstrated very
clearly that foot and mouth disease virus (FMDV) is so highly
contagious that rapid diagnosis is required to control its spread.
See, e.g., Adam, D., Nature 410:398 (2001) and Enserink, M.,
Science 291:2298-2300 (2001).
[0004] Foot and Mouth Disease Virus (FMDV) is actually a group of
closely related viruses, classified as members of the genus
Aphthovirus and family Picornaviridae. The genus Aphthovirus has
two members, FMDV and Equine Rhinitis A Virus (ERV-1). The second
genus member, ERV-1, shares some sequence homology with FMDV, but
is not a cause of foot and mouth disease (FMD). ERV-1 is the agent
of an equine respiratory disease (horses are not susceptible to
FMDV).
[0005] There are seven serotypes of FMDV: types A, O, C, Asia 1,
Sat 1 (South African Territories), Sat 2, and Sat 3. Serotypes are
distinguishable by serotype-specific enzyme linked immunosorbent
assays (ELISA).
[0006] Because of the range of species affected, the high rate of
infectivity, and the fact that FMDV is shed before clinical signs
occur, FMD is one of the most feared reportable diseases known in
North America. Disease caused by FMDV is devastating to farm
animals and can have a major economic impact on countries producing
cloven-hoofed animals (cattle, pigs, sheep, goats and camelids) or
their products. Clearly, new and more sensitive assays for the
detection of this disease are needed.
[0007] A variety of methods for the detection of FMDV have been
developed. These fall into three general categories: 1) detection
of FMDV peptides; 2) detection of FMDV generated antibodies; and 3)
detection of FMDV genetic material.
[0008] A number of peptides have been identified that are unique to
the FMDV and are considered diagnostic for its presence. These
include both structural proteins as well as non-structural proteins
(see, e.g., Yi et al., U.S. Pat. No. 6,048,538; Saeki et al., U.S.
Pat. No. 5,639,601).
[0009] In other cases methods have been developed to detect
antibodies generated by the infected animal to the FMDV. The ELISA
assay is a preferred format (see, e.g., Gilles et al., J.
Virological Methods 107(1):89-98 (2003); Mackay et al., J.
Virological Methods 97(1-2):33-48 (2001); Bergmann et al., Archives
of Virology 145(3):473-489 (2000); and Ferris, N. P., Towards
Livestock Disease Diagnosis and Control in the 21st Century,
Proceedings of an International Symposium on Diagnosis and Control
of Livestock Diseases Using Nuclear and Related Techniques, Vienna,
Apr. 7-11, 1997 (1998), Meeting Date 1997, 65-77, International
Atomic Energy Agency, Vienna, Austria).
[0010] A common and effective method of assay has been the use of
primer directed nucleic amplification methods for the amplification
of diagnostic portions of the FMDV genome. These methods are based
on the isolation of primers or probes that are particularly
diagnostic for the presence of the virus. Collins et al.
(Biochemical and Biophysical Research Communications 297(2):267-274
(2002)) teach an isothermal method of nucleic acid sequence-based
amplification using primers based on a variety of loci in the FMDV
genome. One of the most popular methods for detection is the use of
a method involving reverse transcription followed by polymerase
chain reaction (RT-PCR). As its name implies, the method involves
the synthesis of DNA by reverse transcription and then the
amplification of DNA by PCR. Callahan et al. (WO 02/095074) use
this method for the detection of FMDV using primers derived from
highly conserved regions of the 3D coding region of the genome.
Reid et al. (J. Virological Methods 105(1):67-80 (2002)) teach a
fluorogenic RT-PCR assay using a primer/probe set designed from the
internal ribosomal entry site region of the virus genome that was
capable of detecting all seven serotypes of the FMDV. The
primer-based methods are amenable to a variety of formats and kits
(see, e.g., Callahan et al., J. American Veterinary Medical
Association 220(11): 1636-1642 (2002).
[0011] All of the above methods have been used in the detection of
FMDV. However, tests with reliable breadth of specificity for
"universal" detection of all strains and increased sensitivity,
along with ease and reliability of use, are still needed in an FMDV
assay. Additionally, because of the high gene mutation rate in the
virus, tests directed to different regions of the genome would be
useful. There is a need, therefore, for a highly sensitive assay
for FMDV that broadly detects most strains of the virus, is rapid,
accurate and easily performed.
SUMMARY OF THE INVENTION
[0012] A method for detecting the presence of FMDV in a sample, the
method comprising performing RT-PCR amplification of the sample
using at least one primer pair selected from the group consisting
of SEQ ID NOs:16 and 17, SEQ ID NOs:16 and 18, SEQ ID NOs:16 and
19, and SEQ ID NOs:16 and 20, to produce an RT-PCR amplification
result; and examining the RT-PCR amplification result to detect for
an amplification product of the primer pair, whereby a positive
detection of the amplification product indicates the presence of
FMDV in the sample. Preferably, a melting curve analysis is used to
detect for an amplification product. The method may also comprise a
step of extracting RNA from the sample, preferably prior to the
step of performing RT-PCR amplification of the sample.
[0013] An isolated polynucleotide for detection of FMDV comprising
SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID
NO:20.
[0014] A kit for detection of FMDV, comprising at least one primer
pair selected from the group consisting essentially of SEQ ID
NOs:16 and 17, SEQ ID NOs:16 and 18, SEQ ID NOs:16 and 19, and SEQ
ID NOs:16 and 20; reverse transcriptase; and thermostable DNA
polymerase.
A replication composition for use in performance of RT-PCR,
comprising at least one primer pair selected from the group
consisting essentially of SEQ ID NOs:16 and 17, SEQ ID NOs:16 and
18, SEQ ID NOs:16 and 19, and SEQ ID NOs:16 and 20; reverse
transcriptase; and thermostable DNA polymerase. Preferably, a
replication composition is in the form of a tablet, and a detection
kit comprises a tablet replication composition of the present
application.
BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE DESCRIPTIONS
[0015] FIG. 1 is the DNA sequence of a synthetic FMD target (SEQ ID
NO:21)
[0016] FIG. 2 is a plasmid map showing the synthetic FMD DNA
construct.
[0017] FIGS. 3A-3B show agarose gel electrophoresis results from
RT-PCR reactions. Specifically, FIG. 3A shows RT-PCR amplification
product obtained using primers P2Fwd-10 and P33-4 and using samples
containing serial log dilutions of the synthetic FMD target RNA
from 10.sup.7 copies to 10.sup.1 copies/test. FIG. 3B shows the
RT-PCR amplification product using the P2Fwd-10 and P33-4 primers
with a representative strain from each of the seven FMD viral
serotypes at a starting viral RNA concentration of 10.sup.2 viral
RNA-copies/test.
[0018] FIG. 4 is a composite picture of three agarose
electrophoresis gels showing the RT-PCR amplification products
formed from FMDV serotype O.sub.Taiwan RNA using the P2Fwd-10
primer in combination with three reverse primers P33-4, LJS1 and
LJS2 primers, respectively.
[0019] FIG. 5 is an agarose electrophoresis gel showing the RT-PCR
amplification products formed from the synthetic FMD RNA using the
P2Fwd-10 primer in combination with P33-4 or P33+ primers.
[0020] FIG. 6 shows the process of melting curve analysis in
general. The change in fluorescence of the target DNA is captured
during melting. Mathematical analysis of the negative of the change
of the log of fluorescence divided by the change in temperature
plotted against the temperature results in the graphical peak known
as a melting curve.
[0021] The invention can be more fully understood from the
following detailed description and the accompanying sequence
listing, which form a part of this application.
[0022] The following sequences conform with 37 C.F.R. 1.821-1.825
("Requirements for Patent Applications Containing Nucleotide
Sequences and/or Amino Acid Sequence Disclosures--the Sequence
Rules") and are consistent with World Intellectual Property
Organization (WIPO) Standard ST.25 (1998) and the sequence listing
requirements of the EPO and PCT (Rules 5.2 and 49.5(a-bis), and
Section 208 and Annex C of the Administrative Instructions). The
symbols and format used for nucleotide and amino acid sequence data
comply with the rules set forth in 37 C.F.R. .sctn.1.822.
[0023] SEQ ID NOs:1-13 are linkers for construction of synthetic
FMD DNA.
[0024] SEQ ID NO:14 and SEQ ID NO:15 encodes primers Amplicon 5'
and Amplicon 3'.
[0025] SEQ ID NO:16 is the nucleotide sequence of a 5' Forward
diagnostic primer, P2Fwd-10, which is derived from 3903-3929 bp of
GenBank AF308157.
[0026] SEQ ID NO:17 is the nucleotide sequence of a 3' Reverse
diagnostic primer, P33-4, which binds to 4086-4108 bp of GenBank
AF308157.
[0027] SEQ ID NO:18 is the nucleotide sequence of a 3' Reverse
diagnostic primer, P33+, which binds to 4083-4111 bp of GenBank
AF308157.
[0028] SEQ ID NO:19 is the nucleotide sequence of a 3' Reverse
diagnostic primer, LJS1, which binds to 4460-4489 bp of GenBank
AF308157.
[0029] SEQ ID NO:20 is the nucleotide sequence of a 3' Reverse
diagnostic primer, LJS2, which binds to 4317-4341 bp of GenBank
AF308157.
[0030] SEQ ID NO:21 is the nucleotide sequence of the synthetic FMD
target shown in FIG. 1.
DETAILED DESCRIPTION OF THE INVENTION
[0031] The disclosure of each reference set forth herein is
incorporated by reference in its entirety.
Definitions
[0032] In this disclosure, a number of terms and abbreviations are
used. The following definitions are provided.
[0033] "Polymerase chain reaction" is abbreviated PCR.
[0034] "Foot and Mouth Disease Virus" is abbreviated FMDV.
[0035] "Foot and Mouth Disease" is abbreviated FMD.
[0036] "Reverse transcription followed by polymerase chain
reaction" is abbreviated RT-PCR.
[0037] The term "isolated" refers to materials, such as nucleic
acid molecules and/or proteins, which are substantially free or
otherwise removed from components that normally accompany or
interact with the materials in a naturally occurring environment.
Isolated polynucleotides may be purified from a host cell in which
they naturally occur.
Conventional nucleic acid purification methods known to skilled
artisans may be used to obtain isolated polynucleotides. The term
also embraces recombinant polynucleotides and chemically
synthesized polynucleotides.
[0038] The terms "polynucleotide", "polynucleotide sequence",
"nucleic acid sequence", and "nucleic acid fragment" are used
interchangeably herein. These terms encompass nucleotide sequences
and the like. A polynucleotide may be a polymer of RNA or DNA that
is single- or double-stranded, that optionally contains synthetic,
non-natural or altered nucleotide bases. A polynucleotide in the
form of a polymer of DNA may be comprised of one or more strands of
cDNA, genomic DNA, synthetic DNA, or mixtures thereof.
[0039] The term "amplification product" refers to nucleic acid
fragments produced during a primer-directed amplification reaction.
Typical methods of primer-directed amplification include polymerase
chain reaction (PCR), reverse transcription followed by PCR
(RT-PCR), ligase chain reaction (LCR) or strand displacement
amplification (SDA). If PCR methodology is selected, the
replication composition may comprise the components for nucleic
acid replication, for example: nucleotide triphosphates, two (or
more) primers with appropriate sequences, DNA or RNA polymerase,
buffers, solutes and proteins. These reagents and details
describing procedures for their use in amplifying nucleic acids are
provided in U.S. Pat. No. 4,683,202 (1987, Mullis, et al.) and U.S.
Pat. No. 4,683,195 (1986, Mullis, et al.). If LCR methodology is
selected, then the nucleic acid replication compositions may
comprise, for example: a thermostable ligase (e.g., T. aquaticus
ligase), two sets of adjacent oligonucleotides (wherein one member
of each set is complementary to each of the target strands),
Tris-HCl buffer, KCl, EDTA, NAND, dithiothreitol and salmon sperm
DNA. See, for example, Tabor et al., Proc. Acad. Sci. U.S.A.,
82:1074-1078 (1985)). Additional methods of RNA replication such as
replicative RNA system (Q.beta.-replicase) and DNA dependent
RNA-polymerase promoter systems (T7 RNA polymerase) are also
contemplated.
[0040] The term "reverse transcription followed by polymerase chain
reaction", or "RT-PCR", refers to a sensitive technique for
qualitative or quantitative analysis of gene expression, cloning,
cDNA library construction, probe synthesis, and signal
amplification in in situ hybridizations. The technique consists of
two parts: synthesis of cDNA from RNA by reverse transcription
(RT), and amplification of a specific cDNA by polymerase chain
reaction (PCR). Reverse Transcriptase is an RNA-dependent DNA
polymerase that catalyses the polymerization of nucleotides using
template RNA, DNA, or RNA:DNA hybrids. It is preferred to utilize a
total RNA isolation technique that yields RNA lacking significant
amounts of genomic DNA contamination, since the subsequent PCR
cannot discriminate between cDNA targets synthesized by reverse
transcription and genomic DNA contamination.
[0041] The term "primer" refers to an oligonucleotide (synthetic or
occurring naturally), which is capable of acting as a point of
initiation of nucleic acid synthesis or replication along a
complementary strand when placed under conditions in which
synthesis of a complementary stand is catalyzed by a
polymerase.
[0042] The term "probe" refers to an oligonucleotide (synthetic or
occurring naturally) that is complementary (though not necessarily
fully complementary) to a polynucleotide of interest and forms a
duplexed structure by hybridization with at least one strand of the
polynucleotide of interest.
[0043] The term "replication inhibitor moiety" refers to any atom,
molecule or chemical group that is attached to the 3' terminal
hydroxyl group of an oligonucleotide that will block the initiation
of chain extension for replication of a nucleic acid strand.
Examples include, but are not limited to: 3'-deoxynucleotides
(e.g., cordycepin), dideoxynucleotides, phosphate, ligands (e.g.,
biotin and dinitrophenol), reporter molecules (e.g., fluorescein
and rhodamine), carbon chains (e.g., propanol), a mismatched
nucleotide or polynucleotide, or peptide nucleic acid units. The
term "non-participatory" will refer to the lack of participation of
a probe or primer in a reaction for the amplification of a nucleic
acid molecule. Specifically a non-participatory probe or primer is
one that will not serve as a substrate for, or be extended by, a
DNA or RNA polymerase. A "non-participatory probe" is inherently
incapable of being chain extended by a polymerase. It may or may
not have a replication inhibitor moiety.
[0044] A nucleic acid molecule is "hybridizable" to another nucleic
acid molecule, such as a cDNA, genomic DNA, or RNA, when a single
stranded form of the nucleic acid molecule can anneal to the other
nucleic acid molecule under the appropriate conditions of
temperature and solution ionic strength. Hybridization and washing
conditions are well known and exemplified in Sambrook, J., Fritsch,
E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd
ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y.
(1989), particularly Chapter 11 and Table 11.1 therein (entirely
incorporated herein by reference). The conditions of temperature
and ionic strength determine the "stringency" of the hybridization.
For preliminary screening for homologous nucleic acids, low
stringency hybridization conditions, corresponding to a Tm of
55.degree., can be used, e.g.,5.times.SSC, 0.1% SDS, 0.25% milk,
and no formamide; or 30% formamide,5.times.SSC, 0.5% SDS. Moderate
stringency hybridization conditions correspond to a higher Tm,
e.g., 40% formamide, with5.times. or 6.times. SSC. Hybridization
requires that the two nucleic acids contain complementary
sequences, although depending on the stringency of the
hybridization, mismatches between bases are possible. The
appropriate stringency for hybridizing nucleic acids depends on the
length of the nucleic acids and the degree of complementation,
variables well known in the art. The greater the degree of
similarity or homology between two nucleotide sequences, the
greater the value of Tm for hybrids of nucleic acids having those
sequences. The relative stability (corresponding to higher Tm) of
nucleic acid hybridizations decreases in the following order:
RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100
nucleotides in length, equations for calculating Tm have been
derived (see Sambrook et al., supra, 9.50-9.51). For hybridizations
with shorter nucleic acids, i.e., oligonucleotides, the position of
mismatches becomes more important, and the length of the
oligonucleotide determines its specificity (see Sambrook et al.,
supra, 11.7-11.8). In one preferred embodiment the length for a
hybridizable nucleic acid is at least about 10 nucleotides. More
preferably a minimum length for a hybridizable nucleic acid is at
least about 15 nucleotides; more preferably at least about 20
nucleotides; and most preferably the length is at least 30
nucleotides. Furthermore, the skilled artisan will recognize that
the temperature and wash solution salt concentration may be
adjusted as necessary according to factors such as length of the
probe.
[0045] "Gene" refers to a nucleic acid fragment that expresses a
specific protein, including regulatory sequences preceding (5'
non-coding sequences) and following (3' non-coding sequences) the
coding sequence. "Native gene" refers to a gene as found in nature
with its own regulatory sequences. "Recombinant DNA construct"
refers to any gene that is not a native gene, comprising regulatory
and coding sequences that are not found together in nature.
Accordingly, a recombinant DNA construct may comprise regulatory
sequences and coding sequences that are derived from different
sources, or regulatory sequences and coding sequences derived from
the same source, but arranged in a manner different than that found
in nature. "Endogenous gene" refers to a native gene in its natural
location in the genome of an organism. A "foreign" gene refers to a
gene not normally found in the host organism, but that is
introduced into the host organism by gene transfer. Foreign genes
can comprise native genes inserted into a non-native organism, or
recombinant DNA constructs. A "transgene" is a gene that has been
introduced into the genome by a transformation procedure.
[0046] The term "operably linked" refers to the association of
nucleic acid sequences on a single nucleic acid fragment so that
the function of one is affected by the other. For example, a
promoter is operably linked with a coding sequence when it is
capable of affecting the expression of that coding sequence (i.e.,
that the coding sequence is under the transcriptional control of
the promoter). Coding sequences can be operably linked to
regulatory sequences in sense or antisense orientation.
[0047] The term "expression", as used herein, refers to the
transcription and stable accumulation of sense (mRNA) or antisense
RNA derived from the nucleic acid fragment of the invention.
Expression may also refer to translation of mRNA into a
polypeptide.
[0048] The terms "plasmid", "vector" and "cassette" refer to an
extra chromosomal element often carrying genes which are not part
of the central metabolism of the cell, and usually in the form of
circular double-stranded DNA molecules. Such elements may be
autonomously replicating sequences, genome integrating sequences,
phage or nucleotide sequences, linear or circular, of a single- or
double-stranded DNA or RNA, derived from any source, in which a
number of nucleotide sequences have been joined or recombined into
a unique construction which is capable of introducing a promoter
fragment and DNA sequence for a selected gene product along with
appropriate 3' untranslated sequence into a cell. "Transformation
cassette" refers to a specific vector containing a foreign gene and
having elements in addition to the foreign gene that facilitate
transformation of a particular host cell. "Expression cassette"
refers to a specific vector containing a foreign gene and having
elements in addition to the foreign gene that allow for enhanced
expression of that gene in a foreign host.
[0049] The term "sequence analysis software" refers to any computer
algorithm or software program that is useful for the analysis of
nucleotide or amino acid sequences. "Sequence analysis software"
may be commercially available or independently developed. Typical
sequence analysis software will include but is not limited to the
GCG suite of programs (Wisconsin Package Version 9.0, Genetics
Computer Group (GCG), Madison, Wis.), BLASTP, BLASTN, BLASTX
(Altschul et al., J. Mol. Biol. 215:403-410 (1990), DNASTAR
(DNASTAR, Inc., Madison, Wis.), and Vector NTi version 7.0. Within
the context of this application it will be understood that where
sequence analysis software is used for analysis, that the results
of the analysis will be based on the "default values" of the
program referenced, unless otherwise specified. As used herein
"default values" will mean any set of values or parameters which
originally load with the software when first initialized.
[0050] Standard recombinant DNA and molecular cloning techniques
used here are well known in the art and are described by Sambrook,
J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A
Laboratory Manual, 2.sup.nd ed., Cold Spring Harbor Laboratory:
Cold Spring Harbor, N.Y. (1989) (hereinafter "Maniatis"); and by
Ausubel, F. M. et al., Current Protocols in Molecular Biology,
published by Greene Publishing Assoc. and Wiley-Interscience
(1987).
The Foot and Mouth Disease Virus Genome
[0051] The FMDV genome (approximately 7-8 kB) consists of a single
RNA positive strand encoding four structural proteins termed VP1,
VP2, VP3, and VP4, and at least 10 non-structural proteins. The
non-structural proteins are encoded within sections of the genome
referred to as P2 and P3. These sections can be further divided
into regions 2A, 2B, and 2C, and 3A, 3B, 3C, and 3D, respectively.
Various combinations of these regions encode proteins involved in
viral replication. The principal viral replicase gene is located in
the region known as 3D, which is about 1.5 kB in size.
[0052] Although seven distinct serotypes of FMDV have been
identified to date, variations within each serotype have also been
identified. Portions of many of these better known and studied
variations have been sequenced; additionally, the complete genome
sequence is available for the several serotypes and variations. See
for example:
[0053] 1. Foot-and-mouth disease virus O genomic RNA, isolate
O1Campos, complete genome (Accession No. AJ320488); Pereda, A. J.,
et al. Arch. Virol. 147 (11): 2225-2230 (2002);
[0054] 2. Foot-and-mouth disease virus SAT 2, complete genome
(Accession No. NC003992);
[0055] 3. Foot-and-mouth disease virus C, complete genome
(Accession No.
[0056] NC002554); Baranowski, E., et al., J. Virol. 72 (b 8):
6362-6372 (1998);
[0057] 4. Foot-and-mouth disease virus O strain China/1/99 (Tibet),
complete genome (Accession No. AF506822);
[0058] 5. Foot-and-mouth disease virus C strain C-S8 clone MARLS,
complete genome (Accession No. AF274010); Baranowski, E., et al.
(supra);
[0059] 6. Foot-and-mouth disease virus O, complete genome
(Accession No.
[0060] AF308157); Beard, C. W. and Mason, P. W. J. Virol. 74 (2):
987-991 (2000)).
[0061] Sequence accession numbers are from the GenBank data base at
National Center for Biotechnology Information, National Library of
Medicine, Bldg. 38A, Room 8N-803, Bethesda, Md. 20894.
Identification of Diagnostic Region and Primer Design
[0062] The present invention includes a set of primers useful in a
variety of assay formats for the highly sensitive detection of the
Foot and Mouth Disease Virus (FMDV). As explained further herein,
these primers may also be used as or in the design of hybridization
probes.
[0063] The 2A/2B locus of the FMD genome was selected for primer
design based on the universal homology observed when multiple of
the seven different serotypes were aligned using Vector NTi
alignment tools. Also, the 2A/2B regions are involved in viral
replication. Thus, it was reasonable to predict that these gene
sequences and subsequent proteins would be conserved among the FMD
serotypes, making them attractive targets for a RT-PCR test.
[0064] Preferred primers used herein are those that have homology
to specific regions of the 2A/2B locus (e.g., bp 3864-3917 and
3918-4379 of AF308157) of the FMD and include the forward or 5'
primer as set forth in SEQ ID NO:16 and the three 3' reverse
primers as set forth in SEQ ID NOs:17-18 and 20. One additional
preferred primer is the 3' reverse primer as set forth in SEQ ID
NO:19, which binds to the 2C region (e.g., bp 4380-5333 of
AF308157) The location in the FMDV Serotype O from which each of
SEQ ID NOs:16-20 is derived is shown below in Table 1.
TABLE-US-00001 TABLE 1 Primer sequences diagnostic for FMDV SEQ ID
Location in FMDV Serotype O Primer No. (GenBank AF308157) P2Fwd-10,
Forward 16 3903-3929 P33-4, Reverse 17 4086-4108 P33+, Reverse 18
4083-4111 LJS1, Reverse 19 4460-4489 LJS2, Reverse 20 4317-4341
These primers are broadly useful to detect FDMV infections across a
plurality of serotypes and variations and in FMDV infections
Assay Methods
[0065] SEQ ID NOs:16-20 may be used in a variety of formats for the
detection of FMDV. Most preferred are primer-directed amplification
methods and nucleic acid hybridization methods.
[0066] These methods may be used to detect FMDV in a sample, e.g.,
from an animal, environmental or food source suspected of coming in
contact with the FMDV. The sample and methods of collecting the
sample may include, but are not limited to: swabs from oral and
nasal cavities, body fluids (e.g., blood, blood serum, urine, fecal
material, saliva, cerebrospinal fluid, lymph fluid, amniotic fluid,
peritoneal fluid), tissues (e.g., muscle, skin) or bone samples.
Additionally, air and soil samples may be used.
Primer-Directed Amplification Assay Methods
[0067] In one preferred embodiment, SEQ ID NOs:16-20 may be used as
primers for use in primer-directed nucleic acid amplification for
the detection of the presence of FMDV. A variety of primer-directed
nucleic acid amplification methods are known in the art including
thermal cycling methods (e.g., PCR, RT-PCR, and LCR), as well as
isothermal methods and strand displacement amplification (SDA).
[0068] The preferred method is PCR, and more specifically RT-PCR
for detection of FMDV. Preferred primer pairs are: (i) SEQ ID
NOs:16 and 17; (ii) SEQ ID NOs:16 and 18; (iii) SEQ ID NOs:16 and
19; and (iv) SEQ ID NOs:16 and 20. Most preferred is the primer
pair SEQ ID NOs:16 and 17.
[0069] Typically, in PCR-type amplification techniques, the primers
have different sequences and are not complementary to each other.
Depending on the desired test conditions, the sequences of the
primers should be designed to provide for both efficient and
faithful replication of the target nucleic acid. Methods of PCR
primer design are common and well-known in the art (Thein and
Wallace, "The use of oligonucleotide as specific hybridization
probes in the Diagnosis of Genetic Disorders", In Human Genetic
Diseases: A Practical Approach, K. E. Davis Ed., (1986) pp 33-50;
IRL: Herndon, V A; and Rychlik, W. (1993) In White, B. A. (ed.),
Methods in Molecular Biology, Vol. 15, pp 31-39, PCR Protocols:
Current Methods and Applications. Humania: Totowa, N.J.).
Amplification Conditions
[0070] A skilled person will understand that generally acceptable
RT-PCR conditions may be used for successfully detecting FMDV using
the primers of the instant invention. Depending on the sample to be
tested, complexity of the assay procedure and degree of sensitivity
required, optimization of the RT-PCR conditions may be necessary to
achieve optimal sensitivity and specificity.
[0071] In a preferred embodiment, RT-PCR is performed on a per test
basis as follows (the source of the reagents is set forth in the
Examples section, unless otherwise noted).
[0072] One reaction tube (i e., one test) contains 50 .mu.L of the
following:
[0073] 1. 45 .mu.L of the following:
TABLE-US-00002 Reagent Final Conc. (per 50 .mu.L) PCR Buffer II pH
8.3 1.times. KCl 50 mM Tris-HCl 10 mM MgCl.sub.2 2 mM DNTP 200 or
250 .mu.m Forward primer (e.g. P2Fwd-10) 600 nM Reverse primer
(e.g. P33-4) 2 .mu.M SYBR .RTM. Green 1.times. FastTaq (or HotTaq)
2.5 U Multiscribe reverse transcriptase 1.25 U RNase Inhibitor 20 U
BSA 24 .mu.G DMSO 3.90% Water (to adjust to Final Conc. of the
above)
[0074] 2. 5 .mu.L of the test sample.
[0075] Total=50 .mu.L/test.
[0076] Another preferred embodiment involves the use of certain
reagents in tableted form. One reaction tube (i.e., one test)
contains 50 .mu.L of the following:
[0077] I. One tablet containing:
TABLE-US-00003 Reagent Final Conc. (per 50 .mu.L) Carbowax* 1.86 mM
Trehalose** 360.5 mM dNTP 250 .mu.M Forward Primer (e.g. P2Fwd-10)
720 nM Reverse Primer (e.g. P33-4) 2.4 .mu.M SYBR .RTM. Green
1.times. FastTaq (or HotTaq) 3 U Multiscribe transcriptase 1.5 U
BSA 28.8 .mu.G *from Sigma Aldrich, Catalog #P5413 **from Sigma
Aldrich, Catalog #T9531
[0078] II. 45 .mu.L of the following:
TABLE-US-00004 Reagent Final Conc. (per 50 .mu.L) PCR Buffer II pH
8.3 1.times. KCl 50 mM Tris-HCl 10 mM MgCl.sub.2 2 mM RNase
Inhibitor 20 U DMSO 3.90% Water (to adjust to Final Conc. of the
above)
[0079] III. 5 .mu.L of the test sample.
[0080] Total=50 .mu.L/test.
[0081] Preferred RT-PCR cycling conditions are:
TABLE-US-00005 Temperature (.degree. C.) Time Stage 1: 50 10 min
Stage 2: 95 6 min* Stage 3 (35 cycles) 95 15 sec 71 60 sec Stage 4:
71 5 min *preferably when FastStart is used; preferably 15 min when
HotTaq is used.
Detection
[0082] Primer-directed amplification products can be analyzed using
various methods.
[0083] Homogenous detection refers to a preferred method for the
detection of amplification products where no separation (such as by
gel electrophoresis) of amplification products from template or
primers is necessary. Homogeneous detection is typically
accomplished by measuring the level of fluorescence of the reaction
mixture in the presence of a fluorescent dye.
[0084] In a preferred embodiment, DNA melting curve analysis is
used to carry out homogenous detection, particularly with the
BAX.RTM. System hardware and reagent tablets from Qualicon Inc. The
details of the system are given in U.S. Pat. No. 6,312,930 and PCT
Publication Nos. WO 97/11197 and WO 00/66777, each of which is
hereby incorporated by reference in its entirety.
[0085] Melting curve analysis detects and quantifies double
stranded nucleic acid molecule ("dsDNA" or "target") by monitoring
the fluorescence of the target amplification product ("target
amplicon") during each amplification cycle at selected time
points.
[0086] As is well known to the skilled artisan, the two strands of
a dsDNA separate or melt, when the temperature is higher than its
melting temperature. Melting of a dsDNA molecule is a process, and
under a given solution condition, melting starts at a temperature
(designated T.sub.MS hereinafter), and completes at another
temperature (designated T.sub.ME hereinafter). The familiar term,
T.sub.m, designates the temperature at which melting is 50%
complete.
[0087] A typical PCR cycle involves a denaturing phase where the
target dsDNA is melted, a primer annealing phase where the
temperature optimal for the primers to bind to the
now-single-stranded target, and a chain elongation phase (at a
temperature T.sub.E) where the temperature is optimal for DNA
polymerase to function.
[0088] According to the present invention, T.sub.MS should be
higher than T.sub.E, and T.sub.ME should be lower (often
substantially lower) than the temperature at which the DNA
polymerase is heat-inactivated. Melting characteristics are
effected by the intrinsic properties of a given dsDNA molecule,
such as deoxynucleotide composition and the length of the
dsDNA.
[0089] Intercalating dyes will bind to double stranded DNA. The
dye/dsDNA complex will fluoresce when exposed to the appropriate
excitation wavelength of light, which is dye dependent, and the
intensity of the fluorescence may be proportionate to concentration
of the dsDNA. Methods taking advantage of the use of DNA
intercalating dyes to detect and quantify dsDNA are known in the
art. Many dyes are known and used in the art for these purposes.
The instant methods also take advantage of such relationship.
[0090] An example of such dyes includes intercalating dyes.
Examples of such dyes include, but are not limited to, SYBR
Green-I.RTM., ethidium bromide, propidium iodide, TOTO.RTM.-1
{Quinolinium, 1-1'-[1,3-propanediylbis
[(dimethyliminio)-3,1-propanediyl]]bis[4-[(3-methyl-2(3H)-benzothiazolyli-
dene) methyl]]-, tetraiodide}, and YoPro.RTM. {Quinolinium,
4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(trimethylammonio)propy-
l]-,diiodide}. Most preferred for the instant invention is a
non-asymmetrical cyanide dye such as SYBR Green-I.RTM.,
manufactured by Molecular Probes, Inc. (Eugene, Oreg.).
[0091] Melting curve analysis is achieved by monitoring the change
in fluorescence while the temperature is increased. When the
temperature reaches the T.sub.MS specific for the target amplicon,
the dsDNA begins to denature. When the dsDNA denatures, the
intercalating dye dissociates from the DNA and fluorescence
decreases. Mathematical analysis of the negative of the change of
the log of fluorescence divided by the change in temperature
plotted against the temperature results in the graphical peak known
as a melting curve (See FIG. 6, which illustrates melting curve
analysis in general).
[0092] The data transformation process shown in FIG. 6 involves the
following:
[0093] 1. Interpolate data to get evenly spaced data points
[0094] 2. Take a log of the fluorescence (F)
[0095] 3. Smooth log F
[0096] 4. Calculate -d(log F)/dT
[0097] 5. Reduce data to 11-13 data points spaced one degree apart
(depending on the target organism).
[0098] A positive detection for FMDV results in the appearance of a
melting curve peak as follows:
TABLE-US-00006 Amplicon from Primer Pair: Melting Peak (.degree.
C.) SEQ ID NOs: 16 and 17 83-87 SEQ ID NOs: 16 and 18 Not Yet
Determined SEQ ID NOs: 16 and 19 Not Yet Determined SEQ ID NOs: 16
and 20 Not Yet Determined
[0099] It is believed that the melting point range of 83-87.degree.
C. exists due to the variation of GC/AT content in each serotype
and the variation among topotypes of each serotype.
[0100] The instant homogenous detection method can be used to
detect and quantify target dsDNAs, from which the presence and
level of target organisms can be determined. This method is very
specific and sensitive. The fewest number of target dsDNA
detectable is between one and 10 under typical reaction conditions
and volumes.
[0101] Homogenous detection may be employed to carry out
"real-time" primer-directed nucleic acid amplifications, using
primer pairs of the instant invention (e.g., "real-time" PCR and
"real-time" RT-PCR). Preferred "real-time" methods are set forth in
U.S. Pat. Nos. 6,171,785 and 5,994,056, each of which is hereby
incorporated by reference in its entirety.
[0102] Another detection method is the 5' nuclease detection
method, as set forth in U.S. Pat. Nos. 5,804,375, 5,538,848,
5,487,972, and 5,210,015, each of which is hereby incorporated by
reference in its entirety.
[0103] A variety of other PCR detection methods are known in the
art including standard non-denaturing gel electrophoresis (e.g.,
acrylamide or agarose), denaturing gradient gel electrophoresis,
and temperature gradient gel electrophoresis. Standard
non-denaturing gel electrophoresis is a simple and quick method of
PCR detection, but may not be suitable for all applications.
[0104] Denaturing Gradient Gel Electrophoresis (DGGE) is a
separation method that detects differences in the denaturing
behavior of small DNA fragments (200-700 bp). The principle of the
separation is based on both fragment length and nucleotide
sequence. In fragments that are the same length, a difference as
little as one base pair can be detected. This is in contrast to
non-denaturing gel electrophoresis, where DNA fragments are
separated only by size. This limitation of non-denaturing gel
electrophoresis results because the difference in charge density
between DNA molecules is near neutral and plays little role in
their separation. As the size of the DNA fragment increases, its
velocity through the gel decreases.
[0105] DGGE is primarily used to separate DNA fragments of the same
size based on their denaturing profiles and sequence. Using DGGE,
two strands of a DNA molecule separate, or melt, when heat or a
chemical denaturant is applied. The denaturation of a DNA duplex is
influenced by two factors: 1) the hydrogen bonds formed between
complimentary base pairs (since GC rich regions melt at higher
denaturing conditions than regions that are AT rich); and 2) the
attraction between neighboring bases of the same strand, or
"stacking". Consequently, a DNA molecule may have several melting
domains with each of their individual characteristic denaturing
conditions determined by their nucleotide sequence. DGGE exploits
the fact that otherwise identical DNA molecules having the same
length and DNA sequence, with the exception of only one nucleotide
within a specific denaturing domain, will denature at different
temperatures or Tm. Thus, when the double-stranded (ds) DNA
fragment is electrophoresed through a gradient of increasing
chemical denaturant it begins to denature and undergoes both a
conformational and mobility change. The dsDNA fragment will travel
faster than a denatured single-stranded (ss) DNA fragment, since
the branched structure of the single-stranded moiety of the
molecule becomes entangled in the gel matrix. As, the denaturing
environment increases, the ds DNA fragment will completely
dissociate and mobility of the molecule through the gel is retarded
at the denaturant concentration at which the particular low
denaturing domains of the DNA strand dissociate. In practice, the
electrophoresis is conducted at a constant temperature (around
60.degree. C.) and chemical denaturants are used at concentrations
that will result in 100% of the DNA molecules being denatured
(i.e., 40% formamide and 7M urea). This variable denaturing
gradient is created using a gradient maker, such that the
composition of each DGGE gel gradually changes from 0% denaturant
up to 100% denaturant. Of course, gradients containing a reduced
range of denaturant (e.g., 35% to 60%) may also be poured for
increased separation of DNA.
[0106] The principle used in DGGE can also be applied to a second
method that uses a temperature gradient instead of a chemical
denaturant gradient. This method is known as Temperature Gradient
Gel Electrophoresis (TGGE). This method makes use of a temperature
gradient to induce the conformational change of dsDNA to ssDNA to
separate fragments of equal size with different sequences. As in
DGGE, DNA fragments with different nucleotide sequences will become
immobile at different positions in the gel. Variations in primer
design can be used to advantage in increasing the usefulness of
DGGE for characterization and identification of the PCR products.
These methods and principles of using primer design variations are
described in PCR Technology Principles and Applications, Henry A.
Erlich Ed., M. Stockton Press, NY, pages 71 to 88 (1988).
Instrumentation
[0107] According to a preferred embodiment, the BAX.RTM. System
(DuPont Qualicon, Wilmington, Del.) and melting curve analysis are
used.
Reagents and Kits
[0108] Any suitable nucleic acid replication composition
("replication composition") in any format can be used.
[0109] A typical replication composition for PCR or RT-PCR
amplification may comprise, for example, dATP, dCTP, dGTP, dTTP,
and a suitable polymerase and reverse transcriptase, in conjunction
with target specific primers, and various cofactors modifying
enzyme/primer specificity and activity.
[0110] A preferred replication composition comprises (a) at least
one pair of PCR primers selected from the group consisting of (i)
SEQ ID NOs:16 and 17, (ii) SEQ ID NOs:16 and 18, (iii) SEQ ID
NOs:16 and 19; and (iv) SEQ ID NOs:16 and 20; (b) thermostable DNA
polymerase; and (c) reverse transcriptase.
[0111] If the replication composition is in liquid form, suitable
buffers known in the art may be used (Sambrook, J. et al. 1989,
Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring
Harbor Laboratory Press).
[0112] Alternatively, if the replication composition is contained
in a tablet form, then typical tabletization reagents may be
included such as stabilizers and binding agents. Preferred
tabletization technology is set forth in U.S. Pat. Nos. 4,762,857
and 4,678,812, each of which is hereby incorporated by reference in
its entirety.
[0113] A preferred kit for detection of FMDV comprises (a) at least
one pair of PCR primers selected from the group consisting of (i)
SEQ ID NOs:16 and 17, (ii) SEQ ID NOs:16 and 18, (iii) SEQ ID
NOs:16 and 19;
[0114] and (iv) SEQ ID NOs:16 and 20; (b) thermostable DNA
polymerase; and
[0115] (c) reverse transcriptase.
[0116] A preferred tablet comprises (a) at least one pair of PCR
primers selected from the group consisting of (i) SEQ ID NOs:16 and
17, (ii) SEQ ID NOs:16 and 18, (iii) SEQ ID NOs:16 and 19; and (iv)
SEQ ID NOs:16 and 20; (b) thermostable DNA polymerase; and (c)
reverse transcriptase. Even more preferably, a kit for detection of
FMDV comprises the foregoing preferred tablet.
[0117] In another preferred embodiment, a replication composition
contains an internal positive control. The advantages of an
internal positive control contained within a PCR reaction have been
previously described (U.S. Pat. No. 6,312,930 and PCT Application
No. WO 97/11197, each of which is hereby incorporated by reference
in its entirety, and include: (i) the control may be amplified
using a single primer; (ii) the amount of the control amplification
product is independent of any target DNA or RNA contained in the
sample; (iii) the control DNA can be tableted with other
amplification reagents for ease of use and high degree of
reproducibility in both manual and automated test procedures; (iv)
the control can be used with homogeneous detection, i.e., without
separation of product DNA from reactants; and (v) the internal
control has a melting profile that is distinct from other potential
amplification products in the reaction
[0118] Control DNA will be of appropriate size and base composition
to permit amplification in a primer-directed amplification
reaction. The control DNA sequence may be obtained from the FMDV
genome, or from another source, but must be reproducibly amplified
under the same conditions that permit the amplification of the
target amplification product.
[0119] The control reaction is useful to validate the amplification
reaction. Amplification of the control DNA occurs within the same
reaction tube as the sample that is being tested, and therefore
indicates a successful amplification reaction when samples are
target negative, i.e. no target amplification product is produced.
In order to achieve significant validation of the amplification
reaction a suitable number of copies of the control DNA or RNA must
be included in each amplification reaction.
[0120] In some instances it may be useful to include an additional
negative control replication composition. The negative control
replication composition will contain the same reagents as the
replication composition but without the polymerase. The primary
function of such a control is to monitor spurious background
fluorescence in a homogeneous format when the method employs a
fluorescent means of detection.
Nucleic Acid Hybridization Methods
[0121] Probes particularly useful in nucleic acid hybridization
methods are any of SEQ ID NOs: 16-20 or sequences derived
therefrom.
[0122] The basic components of a nucleic acid hybridization test
include a probe, a sample suspected of containing FMDV, and a
specific hybridization method. Probes are single stranded nucleic
acid sequences which are complementary to the nucleic acid
sequences to be detected. Probes are "hybridizable" to the nucleic
acid sequence to be detected. Typically the probe length can vary
from as few as 5 bases to the full length of the FMDV diagnostic
sequence and will depend upon the specific test to be done. Only
part of the probe molecule need be complementary to the nucleic
acid sequence to be detected. In addition, the complementarity
between the probe and the target sequence need not be perfect.
Hybridization does occur between imperfectly complementary
molecules with the result that a certain fraction of the bases in
the hybridized region are not paired with the proper complementary
base. A probe may be composed of either RNA or DNA. The form of the
nucleic acid probe may be a marked single stranded molecule of just
one polarity or a marked single stranded molecule having both
polarities present. The form of the probe, like its length, will be
determined by the type of hybridization test to be done.
[0123] The sample may or may not contain the FMDV. The sample may
take a variety of forms, however will generally be extracted from
an animal, environmental or food source suspected of coming in
contact with the FMDV. The sample and methods of collecting the
sample may include, but are not limited to: swabs from oral and
nasal cavities, body fluids (e.g., blood, blood serum, urine, fecal
material, saliva, cerebrospinal fluid, lymph fluid, amniotic fluid,
peritoneal fluid), tissues (e.g., muscle, skin) or bone samples.
Additionally, air and soil samples may be used.
[0124] The FMDV RNA may be detected directly but most preferably,
the sample nucleic acid must be made available to contact the probe
before any hybridization of probe and target molecule can occur.
Thus the organism's DNA must be free from the cell and placed under
the proper conditions before hybridization can occur. Methods of in
solution hybridization necessitate the purification of the DNA in
order to be able to obtain hybridization of the sample DNA with the
probe. This has meant that utilization of the in solution method
for detection of target sequences in a sample requires that the
nucleic acids of the sample must first be purified to eliminate
protein, lipids, and other cell components, and then contacted with
the probe under hybridization conditions. Methods for the
purification of the sample nucleic acid are common and well known
in the art (Maniatis, supra).
[0125] Similarly, hybridization methods are well defined. Typically
the probe and sample must be mixed under conditions which will
permit nucleic acid hybridization. This involves contacting the
probe and sample in the presence of an inorganic or organic salt
under the proper concentration and temperature conditions. The
probe and sample nucleic acids must be in contact for a long enough
time that any possible hybridization between the probe and sample
nucleic acid may occur. The concentration of probe or target in the
mixture will determine the time necessary for hybridization to
occur. The higher the probe or target concentration, the shorter
the hybridization incubation time needed.
[0126] In one preferred embodiment, hybridization assays may be
conducted directly on cell lysates, without the need to extract the
nucleic acids. This eliminates several steps from the
sample-handling process and speeds up the assay. To perform such
assays on crude cell lysates, a chaotropic agent is typically added
to the cell lysates prepared as described above. The chaotropic
agent stabilizes nucleic acids by inhibiting nuclease activity.
Furthermore, the chaotropic agent allows sensitive and stringent
hybridization of short oligonucleotide probes to DNA at room
temperature (Van Ness and Chen, Nucl. Acids Res. 19:5143-5151
(1991)). Suitable chaotropic agents include guanidinium chloride,
guanidinium thiocyanate, sodium thiocyanate, lithium
tetrachloroacetate, sodium perchlorate, rubidium
tetrachloroacetate, potassium iodide, and cesium trifluoroacetate,
among others. Typically, the chaotropic agent will be present at a
final concentration of about 3M. If desired, one can add formamide
to the hybridization mixture, typically 30-50% (v/v).
[0127] Alternatively, one can purify the sample nucleic acids prior
to probe hybridization. A variety of methods are known to one of
skill in the art (e.g., phenol-chloroform extraction, IsoQuick
extraction (MicroProbe Corp., Bothell, Wash.), and others).
Pre-hybridization purification is particularly useful for standard
filter hybridization assays. Furthermore, purification facilitates
measures to increase the assay sensitivity by incorporating in
vitro RNA amplification methods such as self-sustained sequence
replication (see for example Fahy et al., In PCR Methods and
Applications, Cold Spring Harbor Laboratory: Cold Spring Harbor,
N.Y. (1991), pp. 25-33) or reverse transcriptase PCR (Kawasaki, In
PCR Protocols: A Guide to Methods and Applications, M. A. Innis et
al., Eds., (1990), pp. 21-27).
[0128] Once the RNA or DNA is released, it can be detected by any
of a variety of methods. However, the most useful embodiments have
at least some characteristics of speed, convenience, sensitivity,
and specificity.
[0129] Various hybridization solutions can be employed. Typically,
these comprise from about 20 to 60% volume, preferably 30%, of a
polar organic solvent. A common hybridization solution employs
about 30-50% v/v formamide, about 0.15 to 1M sodium chloride, about
0.05 to 0.1M buffers, such as sodium citrate, Tris-HCl, PIPES or
HEPES (pH range about 6-9), about 0.05 to 0.2% detergent, such as
sodium dodecylsulfate, or between 0.5-20 mM EDTA, FICOLL (Pharmacia
Inc.) (about 300-500 kilodaltons), polyvinylpyrrolidone (about
250-500 kdal), and serum albumin. Also included in the typical
hybridization solution will be unlabeled carrier nucleic acids from
about 0.1 to 5 mg/mL, fragmented nucleic DNA (e.g., calf thymus or
salmon sperm DNA, or yeast RNA), and optionally from about 0.5 to
2% wt/vol glycine. Other additives may also be included, such as
volume exclusion agents which include a variety of polar
water-soluble or swellable agents (e.g., polyethylene glycol),
anionic polymers (e.g., polyacrylate or polymethylacrylate), and
anionic saccharidic polymers (e.g., dextran sulfate).
[0130] Nucleic acid hybridization is adaptable to a variety of
assay formats. One of the most suitable is the sandwich assay
format. The sandwich assay is particularly adaptable to
hybridization under non-denaturing conditions. A primary component
of a sandwich-type assay is a solid support. The solid support has
adsorbed to it or covalently coupled to it immobilized nucleic acid
probe that is unlabeled and complementary to one portion of the DNA
sequence.
[0131] The sandwich assay may be encompassed in an assay kit. This
kit would include a first component for the collection of samples
from an animal suspected of having contracted the FMDV and buffers
for the disbursement and lysis of the sample. A second component
would include media in either dry or liquid form for the
hybridization of target and probe polynucleotides, as well as for
the removal of undesirable and nonduplexed forms by washing. A
third component includes a solid support (dipstck) upon which is
fixed (or to which is conjugated) unlabeled nucleic acid probe(s)
that is (are) complementary to a part of the FMDV genome. A fourth
component would contain labeled probe that is complementary to a
second and different region of the same DNA strand to which the
immobilized, unlabeled nucleic acid probe of the third component is
hybridized.
[0132] In another preferred embodiment, SEQ ID NOs:16-20 or
derivations thereof may be used as 3' blocked detection probes in
either a homogeneous or heterogeneous assay format. For example, a
probe generated from these sequences may be 3' blocked or
non-participatory and will not be extended by, or participate in, a
nucleic acid amplification reaction. Additionally, the probe
incorporates a label that can serve as a reactive ligand that acts
as a point of attachment for the immobilization of the
probe/analyte hybrid or as a reporter to produce detectable signal.
Accordingly, genomic or cDNA isolated from a sample suspected of
harboring the FMDV is amplified by standard primer-directed
amplification protocols in the presence of an excess of the 3'
blocked detection probe to produce amplification products. Because
the probe is 3' blocked, it does not participate or interfere with
the amplification of the target. After the final amplification
cycle, the detection probe anneals to the relevant portion of the
amplified DNA and the annealed complex is then captured on a
support through the reactive ligand.
[0133] In some instances it is desirable to incorporate a ligand
labeled dNTP, with the label probe in the replication composition
to facilitate immobilization of the RT-PCR reaction product on a
support and then detection of the immobilized product by means of
the labeled probe reagent. For example a biotin, digoxigenin or
digoxin labeled dNTP could be added to RT-PCR reaction composition.
The biotin or digoxin incorporated in the RT-PCR product could then
be immobilized respectively on to a strepavidin, anti-dixogin or
antidigoxigenin antibody support. The immobilized RT-PCR product
could then be detected by the presence of the probe label.
[0134] Probes of the instant invention may be designed in several
alternate forms. The 3' end of the probe is blocked from
participating in a primer extension reaction by the attachment of a
replication inhibiting moiety. Typical replication inhibitor
moieties will include, but are not limited to: dideoxynuleotides,
3-deoxynucleotide, a sequence of mismatched nucleosides or
nucleotides, 3' phosphate groups and chemical agents. Cordycepin
(3' deoxyadenosine) is preferred.
[0135] The replication inhibitor is covalently attached to the 3'
hydroxy group of the 3' terminal nucleotide of the
non-participatory probe during chemical synthesis, using standard
cyanoethyl phosphoramidite chemistry. This process uses solid phase
synthesis chemistry in which the 3' end is covalently attached to
an insoluble support (controlled pore glass, or "CPG") while the
newly synthesized chain grows on the 5' terminus.
3-deoxyribonucleotides are the preferred replication inhibitors.
Cordycepin (3-deoxyadenosine) is most preferred. Since the
cordycepin will be attached to the 3' terminal end of the probe,
the synthesis is initiated from a cordycepin covalently attached to
CPG, 5-dimethoxytrityl-N-benzoyl-3-deoxyadenosine (cordycepin),
2-succinoyl-long chain alkylamino-CPG (Glen Research, Sterling,
Va.). The dimethoxytrityl group is removed and the initiation of
the chain synthesis starts at the deprotected 5' hydroxyl group of
the solid phase cordycepin. After the synthesis is complete, the
oligonucleotide probe is cleaved off the solid support leaving a
free 2' hydroxyl group on the 3'-terminally attached cordycepin.
Other reagents can also be attached to the 3' terminus during the
synthesis of the non-participatory probe to serve as replication
inhibitors. These include, but are not limited to: other
3-deoxyribonucleotides, biotin, dinitrophenol, fluorescein, and
digoxigenin. Each of these reagents are also derivatized on CPG
supports (Glen 5 Research; Clonetech Laboratories, Palo Alto,
Calif.).
EXAMPLES
[0136] The present invention is further defined in the following
Examples. It should be understood that these Examples, while
indicating preferred embodiments of the invention, are given by way
of illustration only. From the above discussion and these Examples,
one skilled in the art can ascertain the essential characteristics
of this invention, and without departing from the spirit and scope
thereof, can make various changes and modifications of the
invention to adapt it to various usages and conditions.
General Methods
[0137] Standard recombinant DNA and molecular cloning techniques
used in the Examples are well known in the art and are described by
Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A
Laboratory Manual; Cold Spring Harbor Laboratory: Cold Spring
Harbor, N.Y. (1989) (Maniatis); by T. J. Silhavy, M. L. Bennan, and
L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor
Laboratory: Cold Spring Harbor, N.Y. (1984); and by Ausubel, F. M.
et al., Current Protocols in Molecular Biology, published by Greene
Publishing Assoc. and Wiley-lnterscience (1987).
[0138] Materials and methods suitable for the maintenance and
growth of bacterial cultures are well known in the art. Techniques
suitable for use in the following examples may be found as set out
in Manual of Methods for General Bacteriology (Phillipp Gerhardt,
R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A.
Wood, Noel R. Krieg and G. Briggs Phillips, eds), American Society
for Microbiology: Washington, D.C. (1994)) or by Thomas D. Brock in
Biotechnology: A Textbook of Industrial Microbiology, 2nd ed.,
Sinauer Associates: Sunderland, Mass. (1989).
[0139] Enzymes and reagents used herein were purchased from the
following vendors: [0140] Applied Biosystems, Foster City, Calif.:
AmpliTaq (Catalog #N808-0160), Multiscribe (Catalog #4311235);
RNase Inhibitor (Catalog #N808-0119); Buffer II (1 mM Tris-HCl pH
8.3, 5 mM KCl) (Catalog #N808-0190); MgCl.sub.2 (Catalog
#N808-0190) [0141] New England Biology, Beverly, Mass.: EcoRl
(Catalog #R0101L); Not l (Catalog #R0189L); T4 DNA Ligase (Catalog
#M0202L); T4 polynucleotide kinase (Catalog #M0201L ) [0142]
Bionexus Inc., Oakland, Calif.: Hot Taq (Catalog #D1002HB); [0143]
Sigma Genosys, The Woodlands, Tex.: Oligonucleotides; [0144]
Qiagen, Valencia, Calif.: Rnase-Free Dnase Set (Catalog #79254);
[0145] Invitrogen Life Technologies, Carlsbad, Calif.: Ampicillian
(Catalog #11593-019); Carbenicillin (Catalog #10177-012); 2%
Agarose E-gels (Cat #G6018-02); Luria Broth (LB) media (Catalog
#10855-021); Triazol LS Reagent (Catalog #10296-028);
Diethylprocarbonate (DEPC) water (Catalog #10813-012) [0146]
Sigma-Aldrich, St. Louis, Mo.: Bovin Serum Albumin (BSA) (Catalog
#A3294); Dimethyl Sulfoxide (DMSO) (Catalog #D8418) [0147] Roche
Diagnostics, Indianapolis, Ind.: FastStart Taq (Catalog #2032937);
dNTP (Catalog #1814362) Additionally, test kits and reagents were
purchased from the following vendors: pCR4-TOPO vector (Invitrogen
Life Technologies, Catalog #45-0030); Qiagen QlAquick PCR
Purification Kit (Qiagen, Catalog #28104); Qiagen Rneasy Mini Kit
(Catalog #74106); Qiagen QlAprep Spin Mini Prep Kit (Catalog #
27106); RNA Transcription kit (Stratagene, Catalog #200340, Cedar
Creek, Tex.); and TOPO TA Cloning Kit Dual Promoter (Invitrogen
Life Technologies, Catalog #45-0640).
[0148] All oligonucleotide primers and linkers were synthesized by
Sigma Genosys Company, The Woodlands, Tex. Polymerase chain
reactions and RNA quantitations were performed using a PTC-225
Peltier Thermal Cycler (M J Research Waltham, Mass.) and GeneQuant
pro (Catalog #80-2110-98; Amersham Pharmacia Biotech, Cambridge,
England).
[0149] Analysis and construction of genetic sequences were
accomplished using the suite of programs available from the
Genetics Computer Group Inc. (Wisconsin Package Version 9.0,
Genetics Computer Group (GCG), Madison, Wis.). Where the GCG
program "Pileup" was used the gap creation default value of 12, and
the gap extension default value of 4 were used. Where the CGC "Gap"
or "Bestfit" programs were used the default gap creation penalty of
50 and the default gap extension penalty of 3 were used. In any
case where GCG program parameters were not prompted for, in these
or any other GCG program, default values were used.
[0150] The meaning of abbreviations is as follows: "sec" means
second(s), "min" means minute(s), "hr" means hour(s), "d" means
day(s), ".mu.L" means microliter(s), "mL" means milliliter(s), "L"
means liter(s), ".mu.M" means micromolar, "mM" means millimolar,
"M" means molar, "mmol" means millimole(s), ".mu.mol" mean
micromole(s), "ng" means nanogram(s), .mu.g" means microgram(s),
"mg" means milligram(s), "g" means gram(s), "mU" means
milliunit(s), and "U" means unit(s).
Construction of a Synthetic RNA Target (3800-4290 bp of FMDV
serotype O)
[0151] A synthetic piece of a foot and mouth virus (FMDV) RNA
serotype O (GenBank Accession Number AF308157; Beard, C. W. and
Mason, P. W., J. Virology 74(2): 987-991 (2000)) was constructed
from base 3800 to 4290. The synthetic FMD target was constructed
using 13 total DNA linkers (SEQ ID NOs: 1-13) comprising both top
and bottom strands (FIG. 1). Notl and EcoRl sites were added to the
sequence of synthetic DNA target to facilitate directional cloning
of the construct behind the T7 promoter in the pCR4-TOPO
vector.
[0152] Linkers were kinased, ligated and PCR amplified using
primers Amplicon 5' and Amplicon 3' (SEQ ID NOs:14 and 15,
respectively) in accordance to published protocols with
modifications (Maniatis, supra, pp 5.68-5.69, 1.68-1.69,
14.2-14.19).
Construction of a Synthetic DNA (3800-4290 bp of FMDV serotype
O)
[0153] To construct the synthetic FMD DNA, linkers (SEQ ID NOs:
1-13) were diluted with DEPC treated water to 25 pmoles/.mu.L.
Linkers (25 pmoles of each) were combined in one tube. To this tube
10 .mu.L of 10.times.T4 Kinase buffer, 100 Units of T4 Kinase, 1 mM
ATP and DEPC water to 100 .mu.L final volume was added. The
reaction was incubated for 30 min at 37.degree. C. The kinased
linkers mix was heated at 95.degree. C. for 20 min in a heat block
to inactivate the kinase and melt all the linkers. After the 20 min
the heat block was turned off and allowed to cool, thereby
facilitating proper linker annealing.
[0154] Once the linkers cooled to room temperature, the ligation
reaction was set-up as follows: in a total volume of 100 .mu.l, 85
.mu.L of the kinased-annealed linkers, 10 .mu.L of 10.times.ligase
buffer, and 50 Units of Ligase were added. The reaction proceeded
for 30 min at room temperature or overnight at 14.degree. C.
Following ligation, the product was amplified by PCR to add
restriction sites (if necessary) and to bulk up the quantity of
product available for subsequent cloning. In a 50 .mu.L reaction 1
.mu.L of annealed, ligated linkers were added to a PCR tube with
1.times. Buffer II, 3.5 mM MgCl.sub.2, 250 .mu.M dNTP, 2.5 Units
Taq, and 20 pmol of forward and reverse primers. Thermocycling
conditions were: 20 cycles of 95.degree. C. (30 sec), 55.degree. C.
(30 sec), 72.degree. C. (30 sec), followed by a final extension at
72.degree. C. (5 min) and a hold at 4.degree. C. The PCR product
was cleaned-up with Qiagen QlAquick PCR Purification Kit. The PCR
product was subsequently digested with Not1EcoRl and cloned into
pCR4-TOPO vector cut with Not1/EcoR1.
Cloning of the Synthetic Target
[0155] The PCR product produced above was cloned using
topoisomerase-cloning technology (TOPO) developed by Invitrogen.
The TOPO TA Cloning Dual Promoter Kit was used for the initial
cloning of the synthetic FMD piece. Putative clones were
transformed into competent E. coli provided by the Invitrogen kit
(Top10F'). E. coli harboring vectors (with or without inserts) were
selected for on LB media containing 50-100 .mu.g/ml ampicillian or
carbenicillian for vector selection. Positive clones, containing
the insert, were determined by growing up individual colonies in 4
ml of LB broth supplemented with 100 .mu.g/ml ampicillian overnight
at 37.degree. C. with 230 rpm shaking. Mini-prep DNA was prepared
using a QlAprep Spin Mini Prep Kit. Clones were analyzed by
restriction endonuclease digest or PCR for correctness, as
determined by insert size.
[0156] The final cloning step entailed removal of the synthetic FMD
fragment by enzymatically removing the insert from the TOPO TA
Cloning vector using Not l and EcoRl. These restriction sites (Notl
and EcoRl) were added to the ends of the synthetic FMD fragment to
facilitate directional cloning of the 5-prime end behind a
prokaryotic T7 promoter of the pCR4-TOPO vector. The T7 promoter
facilitates RNA transcription of the synthetic FMD fragment. The
final synthetic FMD construct (FIG. 2), was sequenced using the M13
forward and reverse primers located on either side of the T7
synthetic FMD portion of the clone; specifically, the M13-20
Forward primer is located at 4437-4452 bp, while the M13 Reverse
primer is located at 629-645 bp. The Synthetic FMD DNA is located
from 36-536 bp and the T7 promoter is located at the 5'-end of the
synthetic FMD DNA from 1-20 bp.
[0157] Sequencing was conducted using fluorescent BigDye terminator
chemistry (Applied Biosystems, Foster City, Calif. 94404). The
synthetic FMD DNA construct had an identical sequence to the
original serotype O sequence from base 3800 to 4290.
[0158] FMD virus is positive strand RNA virus. A positive stand RNA
copy of the synthetic FMD DNA molecule prepared above was
synthesized by copying the FMD DNA (FIG. 1) using a T7 polymerase
and the Stratagene RNA transcription kit. The RNA transcripts
product was then purified and used as a surrogate FMD target
molecule for reverse transcription polymerase chain reaction
(RT-PCR). In this process, the synthetic FMD construct was first
linearized with EcoRl. The digested DNA was passed through a Qiagen
PCR clean-up column, thus facilitating removal of restriction
endonucleases and salts. The T7 polymerase included in Stratagene's
RNA Transcription kit was used to synthesize RNA from the T7
promoter located adjacent to the FMD synthetic construct. Synthetic
RNA was purified using Qiagen's Mini-RNA clean-up protocol
including the optional 15-minute DNAse step. RNA was eluded in DEPC
treated water. Molecules of synthetic FMD RNA per micro liter were
determined spectrophotometrically (GeneQuant pro) and log base ten
serial dilutions were routinely generated for use in RT-PCR
reactions.
Example 1
Demonstration of an RT-PCR Assay for Detection of FMD Using
Synthetic FMDV RNA
[0159] A single step RT-PCR assay for the FMD target sequence was
performed on the synthetic FMD RNA target using the following
reagents and conditions. Each reaction was performed in a 50 .mu.l
total reaction volume.
[0160] First, a pre-reaction mix was prepared for each of the four
primer pairs, as follows. The forward primer P2Fwd-10 (SEQ ID
NO:16) and reverse primer (SEQ ID NO:17, 18, 19, or 20) were
dissolved in water and added respectively to the reaction solution
at concentrations of respectively at 600 nM and 2 .mu.M per test.
Buffer II (1.times.) was added to comprise a final concentration of
1 mM Tris-HCl pH 8.3, 5 mM KCl and 3.5 mM MgCl.sub.2. Nucleotides
were used at 250 .mu.M per test. BSA was used at a final
concentration of 0.6 mM per test. SYBR Green (Catalog #
517695#S7564; Molecular Probes, Eugene, Oreg.) was added in DMSO to
a final dilution of 1:40,000. Enzymes were used at 2.5 Units Taq
polymerase, 20 Units Rnase Inhibitor, and 1.25 Units Multiscribe
reverse transcriptase per 50 .mu.l test. The reaction solution (45
.mu.l) was then stored on ice.
[0161] Samples containing synthetic RNA dissolved in water were
added at 5 .mu.l per reaction. The tube(s) were sealed and then
thermal cycled using the following conditions: [0162] 50.degree. C.
10 minutes (RT step); [0163] 95.degree. C. 15 minutes (Taq
activation step); [0164] 95.degree. C. 15 second (denature step);
[0165] 71.degree. C. 1 minute (anneal and extend step); [0166]
Repeat denature and anneal steps 35 times; [0167] 71.degree. C. 10
minutes; [0168] 4.degree. C. hold.
[0169] The RT-PCR reaction products were then analyzed using
agarose gel electrophoresis using 2% E-gels. Following
electrophoresis the gels were then viewed to determine the presence
or absence of a correct size RT-PCR product (224 bp product of SEQ
ID NOs:16 and 17; P2Fwd-10/LJS1 (SEQ ID NOs:16 and 19) and
P2Fwd-10/LJS2 (SEQ ID NOs:16 and 20) primer sets form larger
products (554 bp and 400 bp, respectively)).
[0170] RT-PCR reactions were performed with each of the four primer
pairs (i.e., SEQ ID NO:16 and each of SEQ ID NOs: 17-20) using
serial log dilutions of the synthetic FMD RNA. Sample
concentrations ranged from 10.sup.7 copies to 10.sup.1
copies/reaction.
[0171] FIG. 3A shows results obtained using the primer pair
P2Fwd-10 and P33-4 (SEQ ID NO:16 and 17). Reactions were carried
out and performed as described above. Specifically, the RT-PCR
product is shown using serial log dilutions of the synthetic FMD
target RNA from 10.sup.7 copies to 10.sup.1 copies/test. As can be
seen in FIG. 3A, the primers sensitivity allows detection of 10
copies of target RNA. The center lane contains molecular weight
markers (Invitrogen low molecular weight standard).
Example 2
RT-PCR Test Response Using FMD Viral Serotypes with P2Fwd-10 and
P33-4 Primer Set
[0172] This example illustrates the RT-PCR assay response to
representative strains of all seven FMD viral serotypes and
demonstrates that all seven serotypes can be detected.
[0173] Virus samples, each containing representative strains of all
seven serotypes of FMD (O, A, C, Asia1, Sat 1, Sat 2 and Sat 3)
were cultivated from field samples using in vitro tissue culture
cell lines by Gordon Ward, USDA, APHIS, Greenport, N.Y. Plaque
forming unit (PFU) and tissue culture infectious dose (TCID.sub.50)
determinations on the cultures established the viral titers for
each sample (as described in Virology, A Practical Approach. B W J
Mahy, Ed.; IRL: Oxford and Washington D.C., 1985; Chapter 2, pp
25-35).
[0174] FMD viral RNA from the samples was isolated using the
Triazol LS extraction chemistry and method as outlined by the
manufacturer (Invitrogen Life Technologies, Catalog #10296-028).
The recovered RNA was then reconstituted in water. Seven log
dilutions were made of each FMD serotype RNA extraction.
[0175] RT-PCR reactions were performed on each of the diluted RNA
serotype samples using the conditions and procedure described in
Example 1. FIG. 3B is a photograph of an agarose electrophoresis
gel showing the typical RT-PCR product formed using samples
containing a 10,000-fold dilution of the original viral RNA
extracts. In this experiment, 5 .mu.l of water was used a Negative,
no-virus sample. P2Fwd-10 and P33-4 primers (SEQ ID NOs:16 and 17)
were used for RT-PCR with a representative strain for each of the
seven FMD viral serotypes at 10.sup.2 viral RNA copies/test. Viral
RNA copies were determined from the viral PFU/ml and TCID.sub.50/ml
culture values. The center lane contains molecular weight markers
(Invitrogen low molecular weight standard). As shown in FIG. 3B,
the correct size RT-PCR product was formed with each FMD viral
serotype demonstrating that the test universally detects RNA from
all seven serotypes.
Example 3
RT-PCR Detection Sensitivity to FMD Serotypes
[0176] The limit of test detection for each of the seven FMD viral
serotypes tested using the RT-PCR assay with the P2Fwd-10/P33-4
primers (SEQ ID NOs:16 and 17) is shown in Table 2. In this
example, serial dilutions of the RNA extracted from the FMD viral
cultures described above were tested using the RT-PCR assay as
described in Example 1. Columns 2 and 4 of the table show the FMD
virus concentrations of the original tissue cultures in
TCDI.sub.50/ml and PFU/ml units. Columns 3 and 4 show the lowest
detectable dose of viral RNA detected by the RT-PCR assay in
TCDI.sub.50/ml and PFU/ml units. As shown below in Table 2, all
seven serotypes of FMD are detectable at levels<10
TCID.sub.50/ml and<0.5 PFU/ml respectively.
TABLE-US-00007 TABLE 2 RT-PCR Test Sensitivity Using
P2Fwd-10-/P33-4 Primer Set RT-PCR RT-PCR FMD Virus Conc.
Sensitivity Virus Conc. Sensitivity Serotype TCID.sub.50/ml
TCID.sub.50/ml PFU/ml PFU/ml O 8.0E + 06 0.8 7.8E + 06 0.02 A
1.0E+06 1 1.2E+06 0.03 C 3.0E+06 3 3.3E+06 0.05 Asia 1 8.0E+06 8
8.0E+06 0.2 Sat1 2.0E+06 2 2.3E+06 0.06 Sat2 3.0E+06 3 3.0E+06 0.08
Sat3 4.0E+06 4 3.7E+06 0.09
Example 4
RT-PCR Assay Using P2Fwd-10 Forward Primer and Three Different
Reverse 3' Primers Forming Larger Products
[0177] Example 4 illustrates the utility of additional primer
combinations to produce RT-PCR test products of different sizes. In
this example, FMD serotype O Taiwan RNA substrate was detected
using the same RT-PCR conditions described in Example 1. However,
in this example, the P2Fwd-10 forward primer (SEQ ID NO: 16) was
used in combination with three different reverse primers: P33-4
(SEQ ID NO:17), LJS1 (SEQ ID NO:19), or LJS2 (SEQ ID NO:20).
[0178] The advantages of the P2Fwd-10/LJS1 and P2Fwd-10/LJS2 primer
sets are that they form a larger product (554 bp and 400 bp,
respectively) compared to P2Fwd-10/P33-4 (224 bp). Also, the
products of P2Fwd-10/LJS1 and P2Fwd-10/LJS2 primer sets can act as
a substrate for half-nested PCR using the P2Fwd-10/P33-4 primer
set.
[0179] Seven 10-fold serial dilutions were prepared of FMD serotype
O RNA extracted in Example 2. These were tested using the above
primer combinations and the RT-PCR reagent concentrations and
thermal cycling conditions in Example 1. Following thermal cycling,
agarose gel electrophoresis was run on the reaction products and
imaged. FIG. 4 illustrates the reaction products formed in response
to RT-PCR reactions using the three primer sets. Specifically, FIG.
4 is a composite picture of three agarose gels showing the RT-PCR
products formed to serotype O .sub.Taiwan RNA using the P2Fwd-10
primer in combination with P33-4, LJS1 and LJS2 primers. The RNA
concentration in PFU/ml used per reaction is listed above each
lane. The reverse primer type and observed product size are listed
to the left of the gel picture. The fourth lane contains the
molecular weight markers (Invitrogen low molecular weight
standard).
[0180] According to the results, each of the primer sets produced
the correct product size as determined by the FMD serotype O gene
sequence. LJS1 and LJS2 primers exhibited test sensitivity down to
10.sup.2 and 10.sup.1 copies, respectively, and P33-4 was sensitive
down to 10.sup.-1 PFU/ml.
Example 5
RT-PCR Test Response Using Various Combinations of 5' Forward and
3' Reverse Primers
[0181] This example illustrates the utility of additional primer
combinations for RT-PCR FMD detection. In this example, serial
dilutions of the synthetic FMD RNA were tested from 10.sup.7 to
10.sup.0 copies per reaction. A negative control was used in
addition to the diluted RNA to determine the response of the test
in the absence of viral RNA. The RNA was amplified with either the
P2Fwd-10/P33-4 (SEQ ID NOs: 16 and 17) or P2Fwd-10/P33+ (SEQ ID
NOs: 16 and 18) primer sets. RT-PCR reactions concentration and
thermal cycling conditions were the same as described in Example 1.
FIG. 5 shows the gel analysis of the reaction products. The RNA
concentration in copies used per reaction is listed above each
lane. The fifth lane contains the molecular weight markers
(Invitrogen low molecular weight) standard). Both primer sets
amplify amplicon RNA. The P2Fwd-10/P33-4 primer set was sensitive
to sample concentrations down to 10.sup.0 copies/reaction and the
P2Fwd-10/P33+ primer set was sensitive down to down to 10.sup.2
copies/reaction.
Sequence CWU 1
1
21186DNAArtificial SequenceLinker for construction of synthetic FMD
DNA 1ggccgcgccc ccggccactt ttggccattc acccgagcga agctagacac
aaacaaaaga 60ttgtggcacc ggtgaaacag cttttg 86284DNAArtificial
SequenceLinker for construction of synthetic FMD DNA 2agctttgacc
tgctcaagtt ggcaggggac gtcgagtcca accctgggcc tttcttcttc 60tctgacgtta
ggtcaaattt ttcc 84387DNAArtificial SequenceLinker for construction
of synthetic FMD DNA 3aagttggttg aaaccatcaa ccagatgcag gaggacatgt
caacaaaaca cggacccgac 60tttaaccggt tggtgtctgc atttgag
87489DNAArtificial SequenceLinker for construction of synthetic FMD
DNA 4gaactggcca ccggagtgaa ggctatcagg accggtctcg atgaggccaa
accctggtac 60aagctcatca agctcttgag ccgcctgtc 89585DNAArtificial
SequenceLinker for construction of synthetic FMD DNA 5atgtatggcc
gctgtagcag cacggtcaaa ggacccagtc cttgtggcca tcatgctggc 60tgacaccggc
cttgagattc tggac 85678DNAArtificial SequenceLinker for construction
of synthetic FMD DNA 6agtacctttg tcgtgaagaa gatctccgac tcgctctcca
gtctctttca cgtaccggcc 60cccgtcttca gtttcggg 78755DNAArtificial
SequenceLinker for construction of synthetic FMD DNA 7cttttgtttg
tgtctagctt cgctcgggtg aatggccaaa agtggccggg ggcgc
55859DNAArtificial SequenceLinker for construction of synthetic FMD
DNA 8acgtcccctg ccaacttgag caggtcaaag ctcaaaagct gtttcaccgg
tgccacaat 59985DNAArtificial SequenceLinker for construction of
synthetic FMD DNA 9ctcctgcatc tggttgatgg tttcaaccaa cttggaaaaa
tttgacctaa cgtcagagaa 60gaagaaaggc ccagggttgg actcg
851086DNAArtificial SequenceLinker for construction of synthetic
FMD DNA 10gtcctgatag ccttcactcc ggtggccagt tcctcaaatg cagacaccaa
ccggttaaag 60tcgggtccgt gttttgttga catgtc 861183DNAArtificial
SequenceLinker for construction of synthetic FMD DNA 11accgtgctgc
tacagcggcc atacatgaca ggcggctcaa gagcttgatg agcttgtacc 60agggtttggc
ctcatcgaga ccg 831285DNAArtificial SequenceLinker for construction
of synthetic FMD DNA 12gagatcttct tcacgacaaa ggtactgtcc agaatctcaa
ggccggtgtc agccagcatg 60atggccacaa ggactgggtc ctttg
851354DNAArtificial SequenceLinker for construction of synthetic
FMD DNA 13aattcccgaa actgaagacg ggggccggta cgtgaaagag actggagagc
gagc 541432DNAArtificial SequencePrimer Amplicon 5' 14gcggccgcgc
ccccggccac ttttggccat tc 321533DNAArtificial SequencePrimer
Amplicon 3' 15gaattcccga aactgaagac gggggccggt acg
331627DNAArtificial SequencePrimer P2-Fwd2, which binds to
3903-3929 bp of GenBank AF308157 16gagtccaacc ctgggccctt cttcttc
271723DNAArtificial SequencePrimer P33-4, which binds to 4086-4108
bp of GenBank AF308157 17atgagcttgt accagggttt ggc
231829DNAArtificial SequencePrimer P33+, which binds to 4083-4111
bp of GenBank AF308157 18ttgatgagct tgtaccaggg tttggcctc
291930DNAArtificial SequencePrimer LJS1, which binds to 4460-4489
bp of GenBank AF308157 19tctgaggcga tccatgcctt aatccagtcg
302025DNAArtificial SequencePrimer LJS2, which binds to 4317-4341
bp of GenBank AF308157 20ggaagaaact cgaggcgacc ttgac
2521516DNAArtificial sequenceSynthetic FMD target 21gcggccgcgc
ccccggccac ttttggccat tcacccgagc gaagctagac acaaacaaaa 60gattgtggca
ccggtgaaac agcttttgag ctttgacctg ctcaagttgg caggggacgt
120cgagtccaac cctgggcctt tcttcttctc tgacgttagg tcaaattttt
ccaagttggt 180tgaaaccatc aaccagatgc aggaggacat gtcaacaaaa
cacggacccg actttaaccg 240gttggtgtct gcatttgagg aactggccac
cggagtgaag gctatcagga ccggtctcga 300tgaggccaaa ccctggtaca
agctcatcaa gctcttgagc cgcctgtcat gtatggccgc 360tgtagcagca
cggtcaaagg acccagtcct tgtggccatc atgctggctg acaccggcct
420tgagattctg gacagtacct ttgtcgtgaa gaagatctcc gactcgctct
ccagtctctt 480tcacgtaccg gcccccgtct tcagtttcgg gaattc 516
* * * * *